Oxygen carriers that are useful, for example, as blood substitutes (sometimes referred to as “oxygen-carrying plasma expanders”) can be grouped into the following three categories: i) perfluorocarbon-based emulsions, ii) liposome-encapsulated hemoglobin, and iii) modified hemoglobin. As discussed below, none has been entirely successful, though products comprising modified cell-free hemoglobin are thought to be the most promising. Perfluorochemical-based emulsions dissolve oxygen as opposed to binding it as a ligand. In order to be used in biological systems, the perfluorochemical must be emulsified with a lipid, typically egg-yolk phospholipid. Though the perfluorocarbon emulsions are inexpensive to manufacture, they do not carry sufficient oxygen at clinically tolerated doses to be effective. Conversely, while liposome-encapsulated hemoglobin has been shown to be effective, it is too costly for widespread use. (See generally, Winslow, Robert M., Hemoglobin-based Red Cell Substitutes, Johns Hopkins University Press, Baltimore, Md. (1992)).
Initial attempts to utilize free hemoglobin from erythrocyte hemolysates as a red cell substitute were unsuccessful. The stromal components were found to be toxic, resulting in coagulopathy and associated renal failure. In 1967, stroma-free hemoglobin solutions had been prepared (Rabiner et al., J. Exp. Med. 126:1127 (1967)). However, stroma-free hemoglobin was found to have a relatively short transfusion half-life of only about 100 minutes
The reason for the short circulation half-life of stroma-free hemoglobin is due to the ability of the protein to disassociate from its tetrameric form into dimers that are rapidly filtered from the circulation by the kidneys. Accordingly, a multitude of methods for cross-linking hemoglobin to prevent dissociation have been devised to minimize the extravasation of hemoglobin. Internally cross-linked hemoglobin (intramolecularly cross-linked) formed by binding amino acid residues between subunits may be achieved with diaspirin (diesters of bis-3,5-dibromosaliocylate, U.S. Pat. No. 4,529,719) or using 2-N-2-formyl-pyridoxal-5′-phosphate and borohydride (Benesch et al., Biochem. Biophys. Res. Commun. 62:1123 (1975)). Intramolecular cross-linking that chemically binds subunits of the tetrameric hemoglobin unit to prevent the formation of dimers is disclosed in U.S. Pat. No. 5,296,465. Simon S. R. and Konigsberg W. H. disclosed the use of bis (N-maleimidomethyl) ether (BME) to generate intramolecular cross-linked hemoglobin (PNAS 56:749-56, (1966)) that was reported to have a four fold increase in half-life when infused into rats and dogs (Bunn, H. F. et al., J. Exp. Med. 129:909-24 (1969)). However, the cross-linking of hemoglobin with BME resulted in the concomitant increase in the oxygen affinity of hemoglobin which at the time was thought to prevent its use as a potential hemoglobin-based oxygen carrier.
Although crosslinked hemoglobins demonstrate longer half lives, these compounds still have a propensity to cause vasoconstriction, which may manifest as hypertension in animals and man (Amberson, W., Science 106: 117-117 (1947); and Keipert, P. et al., Transfusion 33: 701-708, (1993)). Human hemoglobin cross-linked between a chains with bis-dibromosalicyl-fumarate (ααHb) was developed by the U.S. Army as a model red cell substitute, but was abandoned by the Army after demonstration of severe increases in pulmonary and systemic vascular resistance (Hess, J. et al., Blood 78: 356A (1991)). A commercial version of this product was also abandoned after a disappointing Phase III clinical trial (HemAssist™ Baxter International, Inc., Deerfield, Ill.; see Winslow, R. M., Vox sang 79: 1-20 (2000)).
The most commonly advanced explanation for the vasoconstriction produced by cell-free hemoglobin is that it readily binds the endothelium-derived relaxing factor, nitric oxide (NO). Three molecular approaches have been advanced in attempting to overcome the NO binding activity of hemoglobin.
The first approach was utilizing recombinant DNA which attempted to reduce the NO binding of hemoglobin by site-specific mutagenesis of the distal heme pocket (Eich et al., Biochem. 35:6976-83 (1996)). Recombinant hemoglobins with reduced affinity for NO have been produced that are less hypertensive in top-load rat experiments (Doherty, D. H. et al., Nature Biotechnology 16: 672-676 (1998); and Lemon, D. D. et al., Biotech 24: 378 (1996)). However, studies suggest that NO binding may not be the only explanation for the vasoactivity of hemoglobin.
The second approach utilized chemical modification in which the size of the hemoglobin was enhanced through oligomerization, which attempted to reduce or possibly completely inhibit the extravasation of hemoglobin from the vascular space into the interstitial space (Hess J. R. et al., J. Appl. Physiol. 74:1769-78 (1978); Muldoon S. M. et al., J. Lab. Clin. Med. 128:579-83 (1996); Macdonal V. W. et al., Artificial Cells, Blood Substitutes and Immobilization Biotechnology 22:565-75 (1994); Furchgott, R. Ann. Rev. Pharmacol. 24:175-97 (1984); and Kilbourne R. et al., Biochem. Biophys. Res. Commun. 199:155-62 (1994)). Cross-linking stroma-free hemoglobin to form poly-hemoglobin is also described in U.S. Pat. Nos. 4,001,200; and 4,001,401.
The third approach involved surface modification of the hemoglobin. Surface modification of hemoglobin involves covalently attaching macromolecules to the reactive groups on the surface of the hemoglobin, such as dextran (Chang J. E. et al., Can. J. Biochem. 55:398 (1977)), hydroxyethyl starch (DE 2,161,086); gelatin (DE 2,449,885); albumin (DE 2,449,885) and polyethylene glycol (DE 3,026,398; U.S. Pat. Nos. 4,670,417; 4,412,989; and 4,301,144).
It has been found that large, surface modified hemoglobin, such as those modified with polyethylene glycol (PEG), were virtually free of the hypertensive effect, even though their NO binding rates were identical to those of the severely hypertensive ααHb (Rohlfs, R. J. et al., J Biol Chem 273: 12128-12134 (1998)). Furthermore, it was found that PEG-hemoglobin was extraordinarily effective in preventing the consequences of hemorrhage when given as an exchange transfusion prior to hemorrhage (Winslow, R. M. et al., J. Appl. Physiol. 85: 993-1003 (1998)).
Conjugation of PEG to hemoglobin also reduces its antigenicity and extends its half-life in circulation. However, the PEG-conjugation reaction has resulted in dissociation of the hemoglobin tetramer into monomer subunits causing gross hemoglobinuria in exchange-transfused rats receiving PEG-conjugates of hemoglobin monomeric units below 40,000 Daltons (Iwashita and Ajisaka, Organ-Directed Toxicity: Chem. Indicies Mech., Proc. Symp., Brown et al., Eds. Pergamon, Oxford, England pgs 97-101 (1981)). A polyalkylene oxide conjugated hemoglobin having a molecular weight greater than 84,000 Daltons was prepared by Enzon, Inc. (U.S. Pat. No. 5,650,388) that carries 10 copies of PEG-5000 chains linked to hemoglobin at its α and ε-amino groups. This degree of substitution was described as avoiding clinically significant nephrotoxicity associated with hemoglobinuria in mammals.
A variety of linkers and methods have been developed for conjugating macromolecules to hemoglobin. Phenyl-based moieties such as 4-phenylmaleimido or 3-phenylmaleimido have been used successfully to conjugate PEG to hemoglobin (U.S. Pat. No. 5,750,725). However, the use of phenyl groups in therapeutic agents is believed by some to be undesirable.
Polyalkylene oxides that do not contain these phenyl groups were prepared having a succinimidyl functional group for binding free ε-amines available on the surface of the hemoglobin (Larwood and Szoka, J. Labelled Compounds Radiopharm. 21:603-14 (1984)). However, the ester bond formed between the polyalkylene chain and the succinimidyl group was found to be easily hydrolyzed in the living body. To address this issue, polyalkylene oxides were activated to produce urethane linkages with ε-amino groups of hemoglobin, which are less susceptible to hydrolytic degradation (U.S. Pat. No. 5,234,903). Other methods have been utilized that employ thiolation of the ε-amines of hemoglobin for binding polyalkylene oxides having maleimide functional groups (U.S. Pat. No. 6,844,317). The thioester bonds formed between the polyalkylene chain and the maleimido group are less susceptible to degradation (U.S. Patent Application No. 2006/0135753).
Conjugation of hemoglobin to polyalkylene oxides has been performed in both the oxygenated and deoxygenated states. U.S. Pat. No. 6,844,317 describes conjugating hemoglobin in the oxygenated or “R” state to enhance the oxygen affinity of the resultant hemoglobin. This is accomplished by equilibrating the hemoglobin with the atmosphere prior to conjugation. Others describe a deoxygenation step prior to conjugation to diminish the oxygen affinity and increase structural stability to withstand the physical stresses of chemical modification, diafiltration and/or sterile filtration and sterilization (U.S. Pat. No. 5,234,903). For intramolecular cross-linking of hemoglobin, it is suggested that deoxygenating the hemoglobin prior to modification may be required to expose Lys 99 of the alpha chain to the amino group-reactive cross-linking reagent (U.S. Pat. No. 5,234,903).
In all the aforementioned methods for surface modifying hemoglobin, the results in terms of conjugation location, efficiency and ultimate conjugate characteristics have been highly unpredictable. For example, the effects of conjugation conditions on the conformation and surface availability of reactive groups on the hemoglobin molecule, as well as the ability of such reactive groups to form stable bonds with linking groups on macromolecules is highly specific to the reactants and the method, and not easily predicted without significant experimentation. In addition, the effects of surface decoration on the ultimate stability, oxygen affinity, vasoactivity and other performance characteristics of the conjugate must be carefully considered.
Accordingly, improved methods for efficiently producing PAO-Hb conjugates with desirable properties are needed. Such PAO-Hb conjugates will preferentially exhibit a high affinity for oxygen, efficiently deliver oxygen to tissues, not be prematurely excreted from the system, be stable and, unlike whole blood, be stored at room temperature. Such conjugates would eliminate the current need for blood type and cross-matching currently performed for blood transfusions and can be easily and economically manufactured.